Protocol Online logo
Top : Forum Archives: : Protein and Proteomics

sucrose gradient, how to make them - (Sep/03/2007 )

Hi guys, I’ve got to make a sucrose gradient, linear, 5 to 30% but I’ve already tried with the gradient maker and was not that nice. Does anyone of you any idea how to prepare them using a single solution, putting that into the tube and freezing them? I’ve heard this is pretty good technique, but I can’t find any reference in Internet. Thank you very much!!!
(as far as I understood, there are also table indicating the concentration of the solution and the range they reach… )
blink.gif

-dcgbio-

QUOTE (dcgbio @ Sep 3 2007, 06:49 AM)
Hi guys, I’ve got to make a sucrose gradient, linear, 5 to 30% but I’ve already tried with the gradient maker and was not that nice. Does anyone of you any idea how to prepare them using a single solution, putting that into the tube and freezing them? I’ve heard this is pretty good technique, but I can’t find any reference in Internet. Thank you very much!!!
(as far as I understood, there are also table indicating the concentration of the solution and the range they reach… )
blink.gif


the usage of a gradient mixer is the right choice but what went wrong? You need two solutions (5% and 30% sucrose); you should not freeze the gradient

-The Bearer-

The problem is that the gradient makers we got in our lab, are suitable for acrylamide gels, but they are just too big for the sucrose gradient. The flow rate is too high (even using 10µl tips inserted into 200µl tips) and the sucrose is mixing improperly giving strange concentration. Indeed, once you run them, the bands are not forming accordingly to the protocol. I’ve heard that if you need 5to30% gradients, you can just prepare a solution ((5+30):2)) = 17.5% sucrose, then you put it into the tubes and freeze them overnight. The day after you defreeze them (at 4°C or RT) and then you freeze them again at -22°C and after the second de-freezing step you should get around 2.5% to around 30% sucrose gradient formed into the tubes… At least this is the experience in our lab and in the protocols for PSI particle isolation… More I don't know
Thank for any supplemental information!

-dcgbio-

QUOTE (dcgbio @ Sep 3 2007, 11:56 PM)
The problem is that the gradient makers we got in our lab, are suitable for acrylamide gels, but they are just too big for the sucrose gradient. The flow rate is too high (even using 10µl tips inserted into 200µl tips) and the sucrose is mixing improperly giving strange concentration. Indeed, once you run them, the bands are not forming accordingly to the protocol. I’ve heard that if you need 5to30% gradients, you can just prepare a solution ((5+30):2)) = 17.5% sucrose, then you put it into the tubes and freeze them overnight. The day after you defreeze them (at 4°C or RT) and then you freeze them again at -22°C and after the second de-freezing step you should get around 2.5% to around 30% sucrose gradient formed into the tubes… At least this is the experience in our lab and in the protocols for PSI particle isolation… More I don't know
Thank for any supplemental information!


I am sorry I never heard of a freeze-thawing protocol to prepare a sucrose gradient; indeed, a gradient mixer should have no much larger volume than the gradient; there is also the possibility to make a step gradient in f.i. +5% steps

-The Bearer-

You can read a paper published by Naoko Tanese:
Methods: A Companion to Methods in Enzymology 12,224-234 (1997)

-alvaning-